Studies on the binding of S-1 to the troponin-tropomyosin-actin complex suggest that S-1 can bind to actin in at least two different conformations. There is a conformation that binds weakly to actin at an angle postulated to be about 90 degrees and a conformation that binds strongly to actin at about a 45 degree angle. The former conformation only occurs transiently in the cross-bridge cycle when ATP or ADP-Pi is bound to myosin, while the latter conformation occurs in the presence of ADP or absence of nucleotide. A stable analog of the 90 degree weak-binding conformation was obtained by modifying S-1 with the bifunctional thiol reagent, N-N'-p-phenylendimaleimide. In the presence of ATP or PPi, pPDM.S-1 binds to actin as weakly as does S-1 + ATP and this binding is unaffected by troponin-tropomyosin. In the presence of ADP or absence of nucleotide, there is a small, but significant difference between the binding of pPDM.S-1 and the binding of S-1 + ATP to actin. This indicates that by artificially modifying the active site of S-1, the myosin cross-bridge may exist in more than just the two normally occurring conformational states. This may be related to the cross-bridge undergoing a continual rotation on the actin as it changes from the 90 degree to the 45 degree conformation during the cross-bridge cycle. In addition, studies using S-1 cross-linked to actin by the zero length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide, indicate that even though the myosin is cross-linked to actin, it is still able to oscillate between the 90 degree and 45 degree conformations during the actomyosin ATPase cycle. Electron micrographs of the cross-linked actin.S-1 preparation show that the structure of the cross-linked S-1 is very different in the presence and absence of ATP. This provides structural evidence for two different conformations.